Many processes—particularly chemical processes—include steps that are carried out at a specific temperature. The efficacy of a particular step may in fact be dependent on the execution of the step at a specific temperature. It may be necessary to either heat or cool the environment in which the process is being carried out to the desired temperature. Monitoring of the temperature of the environment of a process is necessary so that heating or cooling can be terminated once the desired temperature is reached. Continued monitoring of the environment temperature is required so that the temperature can be maintained at the desired level by heating or cooling as necessary. The same considerations apply when different temperatures are required for further steps in a process.
Means for monitoring temperature are readily available including means for measuring temperature at specific sites within the environment of a process that has temperature-dependent steps. For example, sensors are known which can be affixed to a solid object to continuously monitor the temperature of the object. Probes are also known that can be inserted into a reaction mixture for the continuous monitoring of the temperature of the mixture.
While it is generally easy to monitor the temperature of the overall environment of a process, it is not always easy to monitor the temperature of process microenvironments. This is despite the existence of probes and sensors for localised monitoring of temperature. An example of a process where micro-environment temperature monitoring is desirable, but difficult to achieve with an accuracy of less than one degree Celsius, is a thermal cycling reaction such as for amplification of DNA. The need for micro-environment monitoring of temperature in such a process will now be explained.
DNA can be amplified by the polymerase chain reaction. In this technique, a denatured duplex DNA sample is incubated with a molar excess of two oligonucleotide primers, one being complementary to a first short sequence of the DNA duplex and the other being identical to a second short sequence upstream of the first short sequence (i.e., more 5′ of the first short sequence). Each primer anneals to its complementary sequence and primes the template-dependent synthesis by DNA polymerase of a complementary strand which extends beyond the site of annealing of the other primer through the incorporation of deoxynucleotide triphosphates. Each cycle of denaturation, annealing and synthesis affords an approximate doubling of the amount of target sequence, where the target sequence is defined as the DNA sequence subtended by and including the primers. A cycle is controlled by varying the temperature to permit successive denaturation of complementary strands of duplex DNA, annealing of the primers to their complementary sequences, and primed synthesis of new complementary sequences. The use of a thermostable DNA polymerase obviates the necessity of adding new enzyme for each cycle, thus allowing automation of the DNA amplification process by thermal cycling. Twenty amplification cycles increases the amount of target sequence by approximately one million-fold.
Devices are available for the thermal cycling of multiple samples, typically for the amplification of DNA. A common format of such devices is the inclusion of a block of heat conductive material which has a plurality of channels or cavities therein for receiving vessels—such as microcentrifuge tubes—in which the desired reactions are executed. Monitoring of temperature is relatively easy in such devices since a temperature probe can be associated with the block.
Devices for thermal cycling of reaction mixtures are also known in which the reaction vessels are held in a rotor which is rotated in a controlled temperature environment such as an insulated chamber containing the rotor. Temperature cycling is effected by heating and cooling of the environment. Such a device is disclosed, for example, in International Patent Application No. PCT/AU98/00277 (Publication No. WO 98/49340).
Since reaction mixtures are rotated in the PCT/AU99/00277 device, it is difficult to accurately measure the temperature of a reaction mixture. Regardless of how well the temperature of the rotor environment is controlled, there can be a temperature difference between a reaction mixture and the environment per se. Since accurate temperature control is essential for most thermal cycling reactions, knowledge of the actual temperature of reaction mixtures is important. Compensation can then be made in the environmental temperature management program to give a desired temperature in an actual reaction mixture.
There is thus a need for a means of determining temperature in a micro-environment when it is not possible to use available temperature sensors in connection with the particular micro-environment. With such a means, it would be possible to calibrate temperature control throughout an environment.